| BackgroundThe liver not only is the junction of the endogenous and exogenous lipid metabolic pathways,but also is an important target organ that maintains the balance of lipid synthesis and catabolism in vivo.High energy dietary intake not only induces systemic imbalance of energy metabolic pathway,but also leads to hepatic lipid metabolism disorders,which is contributed to metabolic syndrome,such as obesity,type 2 diabetes,hyperlipidemia and NAFLD.In recent years,NAFLD is a global spread trend,which the incidence is a sharp rise in China,and gradually presenting on the low-age group.The pathological feature of clinical NAFLD is hepatocellular fat ectopic deposition.If lots of fat is diffuse infiltrating,it will evolve into NASH as soon,and even liver fibrosis or cirrhosis.The pathogenesis of NAFLD is increased triglyceride DNL in the liver cells,and decreased lipolysis and lipids transport,resulting in the accumulation of triglycerides,which seriously damage the imbalance of lipid metabolism in hepatocytes.Thus,hepatic lipid metabolism dysregualtion is the basis for fatty liver formation.In present,the clinical mainly takes physical therapy,lipid-lowering drugs and liver protective agents to treat NAFLD.But drugs have little effect,which cannot completely meet the treatment of NAFLD.Therefore,it is imperative to speed up and research the targeted drugs of NAFLD treatment.SIRT1 is a NAD+-dependent deacetylase.Studies found that it not only involves in cellular energy metabolism,but also improves skeletal muscle’s insulin sensitivity,hepatocellular inflammation and oxidative stress.Recent study showed that SIRT1 plays a potent role in the regulation of NAFLD.One example indicated that hepatic triglyceride synthesis and ectopic deposition are increased in normal diet-fed Sirt1+/-mice.Another example showed that SIRT1 overexpression markedly inhibits HFD-induced steatosis.Studies showed that SIRT1 regulates related-nuclear transcription factors involved in lipid synthesis,such as SREBP1 c and PPARγ.Deacetylation of SREBP-1c by SIRT1 in hepatocytes significantly inhibits the transcriptional activity of SREBP-1c,and markedly weakens triglyceride DNL pathway.In the Sirt1-/-mouse model,loss of SIRT1 causes a sharp decrease of adipocytes,and leads to PPARγ inactivity.Subsequently researchers found that SIRT1 directly deacetylates PPARγ cofactor and blocks the differentiation and maturation of adipocytes.In addition,SIRT1 also deacetylates LKB1.It is well-known that deacetylated LKB1 by SIRT1 phosphorylates AMPK.The circadian rhythm exchange of AMPK depends on the regulation of the hypothalamus.Therefore,AMPK is a key energy receptor,which activity is subject to AMP/ATP ratio.AMPK is directly phosphorylated by both of LKB1 and CaMKK.Activated AMPK directly regulates ATGL and HSL expression,and promotes fat mobilization.Phosphorylated ACC by AMPK results in reduced malonyl-CoA production,and then activates CPT-1α,which accelerates fatty acids oxidation and utilization.Taken together,we understand that SIRT1/AMPK pathway plays an important role in regulating lipid homeostasis in hepatocytes,and also provides a new way of NAFLD treatment.Studies confirmed that the pharmacological effects of wolfberry are directly related to the important chemical composition of LBP.LBP has a potent and markedly function in anti-aging,anti-tumor,cell regulation,neuromodulation,immune regulation because of itself antioxidant effect.Our studies previously reported that LBP significantly reduces blood and hepatic TG in HFD-induced NAFLD mice.However,the mechanism by which LBP regulates lipid metabolism remains unclear.As mentioned,the liver is the main organ of fat synthesis and storage.The balance of fatty acid synthesis and oxidation is the most important mechanism of lipid metabolism.How LBP regulates the expression and activity of SIRT1 as key energy regulator;How LBP regulates intracellular lipid metabolism through SIRT1/AMPK signaling pathway? The answer will provide a profound and detailed theoretical basis for best understanding of LBP.AimTo confirm whether LBP may regulate lipid metabolism in hepatocytes through SIRT1;To investigate whether LBP directly regulates the expression and activity of SIRT1 in hepatocytes;To confirm whether LBP improves high-fat-induced NAFLD by activating SIRT1/AMPK pathway.Materials and Methods1.HepG2 cells were incubated with the different concentrations of LBP for 24 h.We checked whether LBP increased intracellular NAD+ level and NAD+/NADH ratio or not.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.SIRT1 recombinant protein and NAD+ substrate were coincubated with different concentrations of LBP for 1 h.We measured whether LBP activated the deacetylation activity of SIRT1.HepG2 cells were treated by LBP at different times.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.2.HepG2 cells were incubated with LBP for 24 h.We checked whether LBP increased deacetylation expression of LKB1 using IP assay.WB analyzed the expressions of phospho-AMPK,phospho-ACC,ATGL and FAS.PA-pretreated HepG2 cells were treated with LBP.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.Hepatic lipid droplets formation was analyzed by Oil red O staining and quantitative.3.SIRT1 siRNA-transfected HepG2 cells were treated with LBP.We checked whether SIRT1 siRNA overturned LBP-mediated LKB1 deacetylation.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.PA-pretreated HepG2 cells were transfected with SIRT1 siRNA,and then treated by LBP for 24 h.WB analyzed the expressions of SIRT1,phospho-AMPK,ATGL and FAS.TG content in hepatocytes was examined by enzyme colorimetry.4.NAFLD induced by HFD was treated with different doses of LBP for 12 weeks.The changes of diet,drinking water,body weight,liver weight and serum TG were detected.Lipid droplets accumulation of liver tissue was observed by H&E and Oil red O staining.Hepatic TG content was measured by enzyme colorimetry.Serum and hepatic malonyl-CoA levels were measured by EILSA kit.QPCR analyzed mRNA levels of ACC1,FAS,ELOVL6,DGAT and CPT-1α.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.5.HepG2 cells were transfected with pCDH-SIRT1 plasmid for 24 h in the absence or presence of PA.WB analyzed the expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS.NAFLD mice were injected with the LV of SIRT1 overexpression by the tail vein.The accumulation of lipid droplets in liver tissue was observed by using H&E and Oil red O staining.Hepatic TG content was measured by enzyme colorimetry.QPCR analyzed mRNA levels of ACC1,FAS,ELOVL6,DGAT and CPT-1α.Serum and hepatic malonyl-CoA levels were analyzed by EILSA kit.The expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS were detected by WB.6.LBP-treatment mice were injected with SIRT1 shRNA LV by the tail vein.The lipid droplets in liver tissue were observed by H&E and Oil red O staining.Hepatic TG content was determined by enzyme colorimetry.The expressions of SIRT1,phospho-AMPK,phospho-ACC,ATGL and FAS were measured by WB.The mRNA expressions of ACC1,FAS,ELOVL6,DGAT and CPT-1α were detected by qPCR.Using EILSA method checked serum and hepatic malonyl-CoA levels.Results1.LBP increased SIRT1 expression in a dose-dependent or time-dependent manner.HepG2 cells were incubated with 0-900 μg/mL LBP for 24 h.LBP increased SIRT1 expression in a dose-dependent manner.HepG2 cells were incubated with 600 μg/mL LBP at 0 h,6 h,12 h,24 h,respectively.LBP increased SIRT1 expression in a time-dependent manner.2.LBP upregulated NAD+/NADH ratio and increased SIRT1 deacetylation activity.HepG2 cells were incubated with 300 or 600 μg/mL LBP for 24 h.We found that LBP increased NAD+ level and NAD+/NADH ratio in a dose-dependent manner.Human recombinant SIRT1 protein and NAD+ were coincubated with 100-600 μg/mL LBP for 1 h.We found that LBP upregulated SIRT1 deacetylation activity in a dose-dependent manner.3.LBP increased LKB1 deacetylation and AMPK phosphorylation via SIRT1.HepG2 cells were incubated with 600 μg/mL LBP for 24 h.IP assay indicated that LBP significantly decreased LKB1 acetylation expression.HepG2 cells were incubated with 0-900 μg/mL LBP for 24 h,respectively.WB showed that LBP significantly increased the expression of phospho-AMPK and phospho-ACC in a dose-dependent manner.HepG2 cells were treated with 250 μM PA for 12 h,and then incubated with 600 μg/mL LBP at different times.The expressions of phospho-AMPK and phospho-ACC were increased by LBP in a time-dependent manner.4.LBP regulated hepatic lipogenesis and lipolysis.HepG2 cells were incubated with 0-900 μg/mL LBP for 24 h.LBP significantly decreased hepatic lipid droplets,markedly increased ATGL expression and decreased FAS expression in a dose-dependent manner.HepG2 cells were treated with 250 μM PA for 12 h,and then incubated with 0-900 μg/mL LBP for 24 h.WB showed that LBP still increased the expression of ATGL and decreased the expression of FAS.5.LBP alleviated NAFLD through SIRT1 / AMPK pathway in vivo.100 or 200 mg/kg LBP significantly reduced body weight and liver weight in mice fed by HFD for 12 weeks.Liver morphology and staining showed that LBP markedly reduced hepatic lipid droplets,and also significantly improved HFD-induced dyslipidemia.WB showed that LBP increased SIRT1,phospho-AMPK,phospho-ACC and ATGL expression and decreased FAS expression.EILSA showed that LBP significantly reduced the accumulation of malonyl-CoA as the ACC product.QPCR showed that LBP inhibited enzyme expressions involved in lipid synthesis pathway and promoted enzyme expression involved in fatty acid oxidation.In addition,LBP improved dyslipidemia and glucose and insulin tolerance test.6.SIRT1 overexpression reduced triglyceride synthesis in hepatocytes.We found that SIRT1 overexpression significantly improved hepatic lipid metabolism,and alleviated HFD-induced NAFLD by in vitro SIRT1-transfected and in vivo SIRT1 LV-injected experiments.7.SIRT1 shRNA reversed the effect of LBP on lipid metabolism.PA-treated HepG2 cells were transfected with SIRT1 siRNA,and then intervented with LBP 24 h.The results showed that SIRT1 siRNA reversed the lipid-lowering effect of LBP.LBP-treated mice fed by HFD were injected with SIRT1 sh RNA LV by the tail vein for 7 days.Historical results showed that SIRT1 shRNA markedly suppressed LBP-lowering lipid effects.WB and qPCR showed that SIRT1 shRNA blocked SIRT1/AMPK signal pathway,and induced increased malonyl-CoA.In addition,SIRT1 shRNA aggravated dyslipidemia and tolerance test.Conclusion 1.LBP increases hepatocellular SIRT1 expression and activity.2.LBP activates SIRT1/AMPK signaling pathway.3.LBP regulates hepatocellular lipogenesis and lipolysis through SIRT1/AMPK pathway. |
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